Background: Though the Lobelia nicotianifolia Roth. is ethnobotanically important plant of India and Sri Lanka its phytoconstituents, antioxidant, and anticancer potential was not yet reported. Objective: The objective of this study is to analyze the phytoconstituents of plant parts of L. nicotianifolia and to determine its antioxidant and cytotoxic potential. Materials and Methods: The plant parts of L. nicotianifolia were extracted with different solvents and qualitative analysis revealed the presence of different phytoconstituents. Total phenolic content (TPC) and total flavonoid content (TFC) were recorded in all plant parts. The extracts were subjected to the antioxidant assays and the potent methanolic extracts were used for cytotoxicity study and further characterized by Fourier–transform infrared spectroscopy and liquid chromatography with a high resolution mass spectrometer (LC–HRMS). Results: The qualitative analysis showed the presence of a wide array of phytoconstituents in L. nicotianifolia plant parts. A significantly higher TPC, TFC, and antioxidant activities were seen in methanolic stem extract. Stem extract showed maximum cytotoxicity against human breast adenocarcinoma (MCF–7) and human cervical adenocarcinoma (HeLa) cell lines whereas, root extract had higher cytotoxicity against human colon adenocarcinoma (HCT–15) cells. The results of cell viability indicated that the methanolic extracts of L. nicotianifolia plant parts exhibited a range of cytotoxic activity in a concentration and time dependent manner against selected cancer cell lines. The LC–HRMS showed the presence of cytotoxic compounds comparatively higher in stem. Conclusion: The study confirms the antioxidant and cytotoxic potential of L. nicotianifolia. To understand the detailed mechanism of cytotoxicity of L. nicotianifolia, it is necessary to study the molecular mechanism involved in this study.